GSM7118836: MM_3 [CUTnRUN]; Homo sapiens; OTHER
4 x 10^6 trypsinized cells were aliquoted and incubated on ice for 10 minutes in 1 ml of nuclear extraction buffer (20 mM HEPES, pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine), the released nuclei were pelleted at 600x g, 5 minutes at 4 oC. A basic wash buffer (WB) consisting of 20 mM HEPES, pH7.5, 150 mM NaCl, 0.05% Triton X-1000, 0.1% BSA, 1x cOmplete proteinase inhibitor, 1x protein phosphatase inhibitor cocktail and 0.5 mM spermidine was used throughout. Between incubation steps, the nuclei were pelleted at 600x g, 3 minutes at 4 oC. Prior to antibody binding, the isolated nuclei were incubated with nutation at 4 oC for 5 minutes in WB containing 2 mM EDTA, followed by nutation at 4 oC for 25 minutes in WB. The nuclei were resuspended in antibody buffer produced by diluting 5 mg of FLAG-M2 in 500 ml of Triton X-100-free WB, and incubated with nutation overnight at 4 oC.  After washing three times in 500 ml of WB on ice (10 minutes each time), the nuclei were incubated at 4 oC for 1 h in 50 ml of WB containing 2.5 ml of EpiCypher pAG-MNase 20x stock (EpiCypher, NC). Washing as above, targeted chromatin cleavage was then conducted by incubating the nuclei on ice for 1 h in 150 ml of BSA-free WB containing 2 mM CaCl2. The cleavage was terminated by adding 150 ml of stop buffer (300 mM NaCl, 20 mM EDTA, 4 mM EGTA and 0.5 ng of E. coli Spike-in DNA (EpiCypher, NC) per 150 ml).  The samples were further incubated with nutation at 4 oC for 1 h, centrifuged at 16,000x g for 5 min at 4 oC and the supernatant was collected. Phenol-chloroform DNA precipitation with 80 mg of glycogen was performed and the Cut&Run-enriched DNA was dissolved in 20 ml of 0.1x TE buffer. Following DNA quantitation by a Qubit fluorometer, Cut&Run DNA libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, MA) per the manufacturer's instruction, with modification in SPRI bead clearance of adaptor ligation and library amplification reactions to retain small-sized DNA fragments (down to ~170 bp in post-library amplification size). The quality of the resultant DNA libraries was confirmed by Agilent TapeStation HS DNA assay (Agilend, CA), and paired-end Illumina NGS was carried out on NovaSeq 6000 with 5 million reads per sample.